Scaffold based binding proteins are becoming legitimate alternatives to antibodies in their ability to bind specific ligand targets. These scaffold binding proteins share the quality of having a stable framework core that can tolerate multiple substitutions in the ligand binding regions. Some scaffold frameworks have immunoglobulin like protein domain architecture with loops extending from a beta sandwich core. A scaffold framework core can be synthetically engineered and used to form a library comprising different sequence variants. The sequence diversity of such libraries is typically concentrated in the exterior surfaces of the proteins such as loop structures or other exterior surfaces that can serve as ligand binding regions.
The fibronectin type III domain (FnIII) has been established as an effective non-antibody “alternative” scaffold for the generation of novel binding proteins. A member of the immunoglobulin superfamily, FnIII has three surface exposed loops at one end of the molecule which are analogous to antibody complementarity determining regions (CDRs). Engineering strategies using this scaffold are based on combinatorial libraries created by diversifying both the length and amino acid sequence of these surface loops. From such libraries, FnIII variants capable of binding to a target of interest can be isolated using various selection methods. The FnIII scaffold offers many advantages compared to conventional antibodies or fragments thereof because it lacks disulfide bonds, can be readily and highly expressed in bacterial systems, and is relatively small. However, a need exists for improved FnIII based polypeptides and methods of producing libraries of such polypeptides.